Neural Differentiation of Dental pulp Stem Cells on Advanced 2D Materials

Objectives: To evaluate the neural differentiation efficiency of dental pulp stem cells (DPSC) on 2D graphene (2DG) and hexagonal boron nitride (hBN) substrates.
Methods: Both 2DG and hBN substrates coated on glass coverslip were characterized using Raman spectroscopy and Atomic force microscopy (AFM). DPSC were seeded on both substrates and cultured in growth media (Control; DMEM+10% FBS) and neural induction media with 40ng/ml bFGF and 20ng/ml EGF for 21 days (treated). Cytocompatability was tested using MTS assay at days 1, 3 and 5. Expression of neural gene namely Nestin, beta III tubulin (TUB-3), and neurofilament-heavy (NF-H) were evaluated by RT-PCR at days 1, 7, 14 and 21. Protein expression for nestin, TUB-3, and NF-H was evaluated after 21 days using flow cytometry. All assays were performed in triplicates and statistical analysis was performed with one way ANOVA and t test (α=0.05).
Results: Raman spectroscopy and AFM showed that the glass surface was completely covered with 2DG. Both the substrates were cytocompatible and supported proliferation of DPSC (p<0.05; 2DG and hBN vs glass). The neural genes expression was higher for cells on 2DG-treated group than glass and hBN treated groups. Moreover cells on 2DG expressed mature neural gene NF-H by day 14, while cells on hBN did not show higher expression than glass treated group even after 21 days. Similarly, the neural proteins expression was higher for cell on 2DG-treated group than glass and hBN treated groups.
Conclusions: The results suggest that both 2DG and hBN were cytocompatible; 2DG substrate supports neural differentiation while hBN does not yield mature neurons by 21 days which is evident by neural genes and proteins expression.